葉綠體運輸機組聯結蛋白M4 (Tic236) 的分析

Abstract

葉綠體在植物中扮演著重要的角色，而葉綠體能否正常運作的關鍵之一在於蛋白質是否能正確地送入至葉綠體內作用。大部分位於葉綠體內的蛋白質都是由細胞核的基因體轉錄，在細胞質轉譯成帶有N端導引訊息的前驅蛋白。在葉綠體的內膜有很多重要的蛋白質，包括代謝產物的轉運蛋白、脂質的生合成酵素、影響葉綠體分裂的組成蛋白及蛋白質運輸機組等，因此這些內膜蛋白質如何正確地插入至葉綠體內膜是重要的研究課題。目前已知有兩種蛋白質插入至葉綠體內膜的路徑: stop-transfer及post-import路徑。使用stop-transfer路徑的蛋白質會先停在葉綠體運輸機組上，之後橫向運輸至內膜。使用post-import路徑的蛋白質會先完全進入至葉綠體基質，形成水溶性的中繼蛋白，再回插至內膜。雖然已知一個內膜蛋白的穿膜區域對於其路徑的選擇有很大的影響，但仍不明瞭是穿膜區域上的什麼特性決定了葉綠體內膜蛋白的路徑選擇。 我們實驗室發現了一個與細菌的運輸機組成員TamB有同源基因關係的葉綠體內膜蛋白質，命名為M4，其功能為連接葉綠體內外膜的運輸機組。本論文首先證實了M4帶有可被切除的導引訊息，我以定點突變實驗估算出M4導引訊息的長度，藉此推算出M4的成熟蛋白大小。之後利用鹼萃取法在豆子及阿拉伯芥葉綠體胞外運輸中，分析M4是走post-import路徑或是stop-transfer路徑插入至內膜。結果證明M4是走stop-transfer路徑插入至葉綠體內膜。之後再將不同葉綠體內膜蛋白的穿膜區域序列做比較，並提出決定葉綠體內膜蛋白插入至內膜使用的路徑的假說。利用在穿膜區域的定點突變，發現走stop-transfer路徑的內膜蛋白的穿膜區域必須夠長才能使蛋白質停在內膜上，而走post-import路徑的內膜蛋白的穿膜區域含有較多側鏈較短的胺基酸。在本篇論文最後，再利用穿透式顯微鏡 (TEM)、螢光顯微鏡及DIC顯微鏡探討若植物M4量降低，會對植物的葉綠體造成什麼影響。結果發現，M4量降低會造成植物細胞中偶爾會有極大的葉綠體出現，而此結果將提供日後研究M4蛋白質在葉綠體中的功能參考依據。

Chloroplasts are essential organelles of plants. Most proteins in chloroplasts are encoded by the nuclear genome and imported from the cytosol. Maintaining chloroplast function is dependent on the correct import and insertion of chloroplast proteins. The inner envelope membrane of chloroplasts contains many important proteins including metabolite transporters, lipid biosynthesis enzymes, components of chloroplast division and protein import machineries. There are two major pathways for proteins insertion into the chloroplast inner membrane: the stop-transfer and the post-import pathways. For proteins that insert through the stop-transfer pathway, their translocation across the inner membrane translocon is halted through their transmembrane domains, which then lateral diffuse into the inner membrane. For proteins that insert through the post-import pathways, they are first fully imported into the stroma to form soluble pathway-intermediates and then reinsert into the inner membrane. Pathway selection is determined by the transmembrane domains but the exact determining features remain unknown. Our lab has newly identified an inner membrane protein we named M4, which is the homolog of the bacterial translocon component TamB. Current data indicate that M4 functions in linking the chloroplast outer and inner membrane translocon complexs. In this thesis, I performed three sets of experiments to characterize M4. I first showed that M4 possesses a cleavable transit peptide and I determined the size of the transit peptide. I then used protein import analyses followed by alkaline extraction, in both pea and an Arabidopsis mutant that retards the reinsertion step of the post-import pathway, to show that M4 uses the stop-transfer pathway for insertion into the inner membrane. I then compared transmembrane domain sequences of inner membrane proteins that are known to insert through one or the other pathway to formulate hypotheses for sequence determinants for pathway selection. The hypotheses were tested using site-directed mutagenesis of the transmembrane domain followed by import assays. The results show that if a chloroplast inner membrane protein is to use the stop-transfer pathway, its transmembrane domain must have a sufficient length to halt the protein at the inner membrane. In the last part of my thesis, I used transmission electron microscope (TEM)、fluorescence microscope and differential interference contrast microscope (DIC) to examine the morphology and size of chloroplasts isolated from two M4 knockdown mutants. My results show that, comparing M4 mutant with the wild type, the general appearance of the chloroplasts and the internal thylakoid membranes are all similar. However extremely large chloroplasts can sometimes be observed in the M4 mutants, suggesting that failure in chloroplast division sometimes occurred in the M4 mutant chloroplasts.